A novel method of rapid detection for heavy metal copper ion via a specific copper chelator bathocuproinedisulfonic acid disodium salt

The extensive usage and production of copper may lead to toxic effects in organisms due to its accumulation in the environment. Traditional methods for copper detection are time consuming and infeasible for field usage. It is necessary to discover a real-time, rapid and economical method for detecting copper to ensure human health and environmental safety. Here we developed a colorimetric paper strip method and optimized spectrum method for rapid detection of copper ion based on the specific copper chelator bathocuproinedisulfonic acid disodium salt (BCS). Both biological assays and chemical methods verified the specificity of BCS for copper. The optimized reaction conditions were 50 mM Tris–HCl pH 7.4, 200 µM BCS, 1 mM ascorbate and less than 50 µM copper. The detection limit of the copper paper strip test was 0.5 mg/L by direct visual observation and the detection time was less than 1 min. The detection results of grape, peach, apple, spinach and cabbage by the optimized spectrum method were 0.91 μg/g, 0.87 μg/g, 0.19 μg/g, 1.37 μg/g and 0.39 μg/g, respectively. The paper strip assays showed that the copper contents of grape, peach, apple, spinach and cabbage were 0.8 mg/L, 0.9 mg/L, 0.2 mg/L, 1.3 mg/L and 0.5 mg/L, respectively. These results correlated well with those determined by inductively coupled plasma-mass spectrometry (ICP-MS). The visual detection limit of the paper strip based on Cu-BCS-AgNPs was 0.06 mg/L. Our study demonstrates the potential for on-site, rapid and cost-effective copper monitoring of foods and the environment.


Materials and methods
All chemical reagents were obtained from Sigma-Aldrich (China), except special instructions.
Yeast strains, culture media, and growth assays. A haploid control yeast S. cerevisiae strain, BY4741, and knock out mutants 27 were purchased from Open Biosystems. ace1Δ, pca1Δ, ftr1Δ and fet3Δ and wild type strains were used to test the sensitivities of Cu + , Cd 2+ , Ni 2+ , Pb 2+ , Cr 3+ and Hg 2+ , respectively. Yeast cells were cultured in synthetic complete media (SC) and 1.5% agar was supplemented into the liquid media for solid medium plates. Yeast strains were cultured at 30 °C. For yeast cell growth assays 28,29 , WT or knock out cells were grown over-night in SC media and re-inoculated (OD 600 = 0.2) into fresh media, and grown to mid-log phase (OD 600 = 0.8-1.0). After dilution to OD 600 = 0.1 and 3 × serial dilutions in sterilized water, ~ 5 µL of cells were spotted on SC plates supplemented with various amounts of CuSO 4 , CdCl 2 , NiSO 4 , CrCl 3 , Hg(NO 3 ) 2 and Pb(NO 3 ) 2 with or without BCS or the non-specific metal chelator EDTA. Cells were grown at 30 °C for 2-3 days prior to photography. Each assay was repeated at least three times using three different colonies to confirm results.

Preparation of standard curve.
To quantify the BCS-Cu + color on paper, the optimized reaction condition (50 mM Tris-HCl pH 7.4, 200 μΜ BCS, 1 mM ascorbate) was used to make a standard curve. A series of CuSO 4 concentrations (0, 0.2, 0.5, 1. 2. 5, 10, 20 mg/L) were added into the reaction system and dripped on paper (0.5 cm × 6 cm of standard filter paper). The reaction solutions were then measured at 490 nm by spectrophotometry.
Sample preparation for color reaction and ICP-MS measurement. 0.1 g sample (grape, peach, apple, spinach, cabbage) was collected and smashed into juice 30 . The juice samples were dissolved in 50 μL 10% nitric acid at room temperature for 20 min and subsequently the whole solution was taken into the reaction system for 490 nm measurement and paper test respectively.
For ICP-MS measurement, the juice samples were dissolved in 70% nitric acid at 70 °C for 3 h and then overnight at room temperature and subsequently diluted in 10% nitric acid. ICP-MS (Agilent Model 7500cs, Santa Clara, CA) was used to quantify metal ions. Metal ion contents were normalized to sample weight 31 Characterization of BCS-AgNPs. The naked eye observation revealed a light yellow color, indicating the formation of BCS-AgNPs. This was further confirmed by conducting UV full-wavelength scanning with a 1.0 cm quartz cell using a UV spectrometer. The absorption spectrum of AgNPs exhibited a distinct peak at 390 nm. The peak shifted to 410 nm and a characteristic absorption peak of BCS-Cu was formed at 490 nm after BCS addition. The ratio of A490 nm/A410 nm served as a parameter for the quantitative detection of Cu + . 10 μL AgNPs solution was placed on a carbon-coated copper grid (300 mesh) for transmission electron microscopy (TEM, JEM-2100) to observe the morphology of the silver nanoparticles in different systems and a particle size distribution map was prepared. The BCS-AgNPs solution was placed in a refrigerated vacuum dryer for 48 h. The dried material was subjected to FTIR (FTIR-7600) analysis in KBr particles within the range of 500-4000 cm -1 .

Statistical analysis.
Descriptive analyses were presented as the mean ± S.D. and statistical comparisons of control and experimental groups were performed using Student's t-test. p < 0.05 was considered to be significant.
Ethics guideline statement. The collection of plant material, comply with relevant institutional, national, and international guidelines and legislation.

Results
A novel biological assay to test BCS binding specificity. Heavy metal ion chelators with high specificity, low toxicity, strong binding ability and color reaction capability are essential to preparing paper test strips of practical use in the detection of trace heavy metal ions. Therefore, screening methods are important to discover new chelators that meet our requirements. In this study, we developed a biological assay based on yeast mutants' sensitivities to heavy metal ions. To determine the specificity of BCS for copper, we assessed whether metal-induced toxicity of several metal-sensitive yeast knockout strains could be recovered by BCS. A nonspecific metal chelator EDTA was used as a positive control. ace1Δ, pca1Δ, ftr1Δ, fet3Δ, ftr1Δ and wild type cell strains were used to test the sensitivities of Cu + , Cd 2+ , Ni 2+ , Pb 2+ , Cr 3+ and Hg 2+ , respectively. WT and knock out mutants were cultured to mid-log phase. Cells were spotted on SC plates supplemented with various amounts of CuSO 4 , CdCl 2 , NiSO 4 , CrCl 3 , Hg(NO 3 ) 2 and Pb(NO 3 ) 2 with or without BCS or EDTA. As expected, ace1Δ cells were highly sensitive to CuSO 4 at 20 μΜ and this growth defect was rescued by supplementation of BCS or EDTA (Fig. 1A). The growth defects induced by other metals, when treated at toxic concentrations to their respective metal-sensitive yeast strains, were not recovered by BCS, but only by EDTA ( Fig. 1B-F). These data indicate that metal chelation by BCS is specific to copper, but not other metal ions. The recovery of cell growth under all metal treatment conditions by positive control EDTA demonstrated that growth defects in this assay were indeed due to metal toxicity.
Identification of optimal BCS-Cu + reaction conditions. A previous report showed that BCS binds Cu (I) in a 2:1 ratio ( Fig. 2A) 34 . The BCS-Cu + complex was scanned by spectrophotometer and the maximum absorbance was found at 490 nm ( Fig. 2B). To optimize the BCS-Cu + reaction conditions, we tested the effects of ascorbate, BCS, CuSO 4 and pH. The 490 nm absorbance was highest when the ascorbate concentration was increased to 0.25-1 mM under the conditions of 25 μM CuSO 4 and 200 μM BCS (Fig. 2C). For the BCS effect, 100-200 μM BCS was optimal to bind with 25 μM CuSO 4 (Fig. 2D). The absorbance was proportional to the concentration of copper in the presence of sufficient BCS and ascorbate (Fig. 2E). There was minimal effect of pH on the absorbance from pH 6.5-8.5 (Fig. 2F). Based on these results, we chose an optimal reaction system for BCS-Cu + binding to consist of 1 mM ascorbate, 200 μM BCS and pH 7.4.

Chemical identification of BCS binding specificity by spectrophotometer.
We developed a biological assay and demonstrated BCS exhibits a strong specificity to copper. To further confirm this finding and the new biological assay, a chemical identification by spectrophotometer was employed to verify BCS binding specificity. The reaction system was 50 mM Tris-HCl pH 7.4, 200 μM BCS, 1 mM ascorbate and different concentrations of metal ions. The reaction system was mixed with CuSO 4 , CdCl 2 , CrCl 3 , Hg(NO 3 ) 2 , NiCl 2 and Pb(NO 3 ) 2 at concentrations from 0-20 mg/L, and absorbance was monitored. We were able to follow the change in absorbance at 490 nm in line with the introduction of copper (Fig. 3A). The other metal ions, including Cd 2+ (Fig. 3B), Cr 3+ (Fig. 3C), Hg 2+ (Fig. 3D), Ni 2+ (Fig. 3E) and Pb 2+ (Fig. 3F) were not able to affect the absorbance at 490 nm. These results were consistent with those of the biological assays. Therefore, chemical identification further confirmed that BCS exhibits a strong specificity to copper and demonstrated the accuracy and validity of biological assay.
Spectrum method and fabrication of a paper strip for the detection of copper. The BCS-Cu + complex generated absorption intensity at 490 nm in proportion to copper content by spectrophotometry [19][20][21] .
In order to accurately obtain the relationship between the reaction chroma and the concentration of copper,   (Fig. 4A). The solution color deepened as the copper concentration increased. The color of solution as low as 0.5 mg/L changed significantly relative to the control color even when judged by naked eyes (Fig. 4A). The solutions were measured by spectrophotometer at 490 nm and a standard curve (y = 0.0192x + 0.0023, R 2 = 0.9993) of copper and absorbance was established (Fig. 4B). The paper strips (0.5 cm × 6 cm) were soaked into these solutions and dried one minute for color display (Fig. 4C). The color of the test strip gradually deepened as the concentration of copper increased (Fig. 4C). In fact, the color of 0.2 mg/L paper still showed change although it was not very obvious (Fig. 4C). These results demonstrated a novel colorimetric paper strip method with a 0.5 mg/L detection limit (food copper detection limit ≤ 10 mg/kg, GB15199-94) and an optimized spectrum method are successfully established for rapid detection of copper ion.
Copper measurements of fruits and vegetables by paper strip and optimized spectrum based on BCS-Cu + color reaction. To test the application of paper strip detection and the optimized spectrum method, grape, peach, apple, spinach and cabbage were purchased from the market. 0.1 g of each sample were smashed and dissolved in 50 μL 10% nitric acid at room temperature for 20 min and subsequently the whole solution was taken into the reaction system for color development, 490 nm measurement and paper test. Compared with the blank control, the five sample solutions displayed obvious color changes (Fig. 5A). The paper strip www.nature.com/scientificreports/ assays showed that the copper contents of grape, peach, apple, spinach and cabbage were 0.8 mg/L, 0.9 mg/L, 0.2 mg/L, 1.3 mg/L and 0.5 mg/L, respectively (Fig. 5B). The detection results of grape, peach, apple, spinach and cabbage by the optimized spectrum method at 490 nm were 0.91 μg/g, 0.87 μg/g, 0.19 μg/g, 1.37 μg/g and 0.39 μg/g, respectively (Fig. 5C). The ICP-MS measurements showed that the copper contents of grape, peach, apple, spinach and cabbage were 0.84 mg/L, 1.07 mg/L, 0.15 mg/L, 0.88 mg/L and 0.23 mg/L, respectively (Fig. 5D). The results of the optimized spectrum method and paper strip assay correlated well with those determined by ICP-MS. Collectively, our results indicated both the optimized spectrum method and paper strip assay were able to reliably quantitate copper in foods.

Characteristics and properties of BCS-AgNPs.
Given that nanomaterial is an effective approach to improve detection sensitivity, Ag nanoparticles (AgNPs) was used to increase the color change of standard paper. BCS possesses sulfonic acid groups, which can be easily bound to AgNPs (Fig. 6A). We prepared BCSfunctionalized silver nanoparticles and determined characteristics and properties. FTIR spectra of BCS, AgNPs and BCS-AgNPs showed that the characteristic peaks of 620 cm -1 and 1190 cm -1 belonged to BCS, and the 1384 cm -1 belonged to AgNPs (Fig. 6B). The 1384 cm -1 peak disappeared after BCS was added. The results indicated that BCS was successfully modified to the surface of AgNPs. TEM images of AgNPs (Fig. 6C), BCS-AgNPs (Fig. 6D) and Cu-BCS-AgNPs (Fig. 6E) revealed the particles were a spheroidal shape. The nano-silver distribution slightly dispersed after the addition of BCS but the aggregation intensified after the addition of copper. The particle size distributions of AgNPs was examined by TEM. Most primary particles were sized within 9-30 nm and the average diameter of particles was ~ 15 nm (Fig. 6F-H). Meanwhile, we optimized Cu-BCS-AgNPs binding conditions and determined the optimal AgNPs:BCS ratio was 2:1 and the optimal reaction time was less than www.nature.com/scientificreports/ 1 s (Fig. 6I-K). Overall, these results suggested that Cu-BCS-AgNPs are very stable in a nano-particle form and display a special color.

Preparation of standard paper strips based on Cu-BCS-AgNPs. In view of Cu-BCS-AgNPs dis-
played excellent properties of aggregation, color development and stability, we next tested the specificity of Cu-BCS-AgNPs. Thirteen element ions, including Cr 3+ , Hg 2+ , Ni 2+ , Pb 2+ , Cd 2+ , Al 3+ , Fe 3+ , Ca 2+ , Na + , Mg 2+ , K + , Zn 2+ and Mn 2+ , were tested using the same BCS-AgNPs system. As shown in Fig. 7A, only Cu + , but not other metal  www.nature.com/scientificreports/ ions, resulted in a significant color change (Fig. 7A). In order to evaluate the interference of other metal ions on copper, the 13 metal ions were mixed with the same concentration Cu + and added into the BCS-AgNPs reaction system. The color changes indicated that other metal ions displayed very weak influence on Cu + binding to BCS-AgNPs (Fig. 7B). In order to accurately obtain the relationship between the reaction chroma and the Cu-BCS-AgNPs, the optimal reaction system was mixed with different concentrations of CuSO 4 from 0-6 mg/L (Fig. 7C). The solution color deepened as the copper concentration increased. The solution color of 0.06 mg/L changed significantly relative to the control color even when judged by naked eyes (Fig. 7C). The solutions were measured by spectrophotometer at 490 nm/410 nm and a standard curve (y = 0.0303x + 0.1353, R 2 = 0.9927) of copper and absorbance ratio was established (Fig. 7D). The paper strips (0.5 cm × 0.5 cm) were soaked into these solutions and dried one minute for color display (Fig. 7E). The color of the test strip gradually deepened as the concentration of copper increased (Fig. 7E). Consistent with color changes of solutions, the color of 0.06 mg/L paper still showed an obvious change by eye (Fig. 7E). These results demonstrated we have successful developed a sensitive colorimetric paper strip method based on Cu-BCS-AgNPs with a 0.06 mg/L detection limit.

Discussion
As an essential element of organisms and a widely used industrial raw material, copper is extensively distributed in water, soil and agricultural products 4,35,36 . Therefore, it is necessary to develop rapid and effective methods for the detection of copper contents in the environment and food to ensure environmental safety and human health. Among all the rapid detection methods for copper ions, paper-based analytical devices (PADs) are becoming a popular research field owing to their portability, on-site detection, simple operation, low cost and multifunctional analysis [10][11][12] . The mechanisms of these paper analytical test strips mainly utilize the combinations of heavy metal ions and chelators to produce color reactions. Hence, chelators possessing high specificity, low toxicity, strong binding ability and ability to produce a color reaction are crucial to preparing paper test strips. BCS has been employed as a chelator in many fields, such as evaluation of the copper (II) reduction assay 19 , determination of D-glucose and D-galactose levels 37 , development of multi-walled carbon nanotube Cu sensors 18 , and quantitation of Cu and Cu-protein complexes 17,18,21 . During our study of copper ion metabolism, we identified the BCS-Cu + complex showed different intensities of yellow on paper strip and in solution proportional to copper levels. Based on this discovery, we developed a rapid, on-site, low-cost and safe single paper strip detection method for copper and this novel method was applied to detect copper levels of fruits and vegetables.
Chelator specificity is key to the practical application of paper strip detection. BCS has been used as a copper chelator for a long time, but there was no intuitive way to reflect its specificity 17 . In this study, we took advantage of heavy metal sensitive yeast mutants to identify the binding capacity of BCS to various heavy metals. This method intuitively showed that BCS displayed strong binding capacity to copper ions but not other heavy metal ions.
To thoroughly compare our method with some other reported methods, we analyzed their features in terms of sensitivity, specificity, detection time, instrument aids, detection object and cost (Table 1). Overall, our method displayed high sensitivity, easy portability, on-site detection, simple operation, low cost and practical applicability.
Although the detection results of grape, peach, apple, spinach and cabbage by the optimized spectrum method and paper strip assays correlated well with those determined by ICP-MS, there are still some aspects worth improving. The first is the sample pretreatment. Concentrated acid digestion is the most common pretreatment for food materials and concentrated HNO 3 is preferentially used owing to its high purity and broad scope of oxidation ability 38 . Despite the high digestion efficiency of concentrated HNO 3 , a high residual acidity and the production of highly acidic digests do not comply with green analytical chemistry requirements 39,40 . To address these shortcomings, 10% diluted HNO 3 was employed to digest in smashed samples for copper analysis. These results indicated that this is a feasible method to completely digest samples of fruits and vegetables, such as grape, peach, apple, spinach and cabbage. But if we desire to expand the application scope of these novel paper strip and optimized spectrum methods to less digestible samples, such as rice, a more effective strategy must be developed. Recently, a microwave-assisted digestion method using diluted HNO 3 was developed for determination of heavy  39 . Lee et al. reported that a diluted nitric acid and hydrogen peroxide mixture is another way to optimize acidic digestion while minimizing environmental impact 40 .
The second aspect to further improve is the detection limit, specifically, to enhance the color intensity under low copper levels. Nanomaterials with unique chemical and electrochemical properties show extensive applications in increasing detection sensitivity 41,42 . Borah et al. reported that GA-AuNP@Tollens' complex as a highly sensitive plasmonic nanosensor strengthened the detection of formaldehyde and benzaldehyde in preserved food products 43 . The use of novel nanomaterials with high signal strength is considered as the most effective strategy to improve the detection limit of the BCS-Cu + based paper strip test but these nanomaterials may increase environmental impact and cost. In this study, Ag nanoparticles (AgNPs) was used to improve the color change of standard paper and increase detection sensitivity. We developed a sensitive colorimetric paper strip method based on Cu-BCS-AgNPs with a 0.06 mg/L visual detection limit.
The third aspect worth improving is the chromatography paper quality. In this research, even the use of common filter paper to display the color change still yielded reasonable detection. Different filter papers may be tried to enhance color quality of the BCS-Cu + complex.
To avoid subjectivity in color judgment, a smartphone can be used as a detector for color judgment albeit with the increase in detection cost 11 . However, if subsequent studies improve the quality of the test paper, it may be possible to accurately judge the color change with the naked eye, similar to the pH paper test strip, and therefore it would not be necessary to use additional equipment to assist color judgment.
In conclusion, the BCS-Cu + complex displayed different intensities of yellow proportional to copper levels in our reaction solution and on paper strip, and this reaction was harnessed to develop a rapid, on-site, low-cost and safe single paper strip test for copper detection which was successfully applied to determine copper contents of fruits and vegetables.

Data availability
The data underlying this article are available from the corresponding author upon request.